Pegylated interleukin-10

ABSTRACT

Interleukin-10 (IL-10) conjugated via a linker to one or more polyethylene glycol (PEG) molecules at a single amino acid residue of the IL-10, and a method for preparing the same, are provided. The method produces a stable mono-pegylated IL-10, which retains IL-10 activity, where pegylation is selective for the N-terminus on one subunit of IL-10 with little or no formation of monomeric IL-10. The method also provides a substantially homogenous population of mono-PEG-IL-10.

This application is a Continuation of co-pending U.S. patent applicationSer. No. 12/200,486, filed Aug. 28, 2008, which is a Continuation ofU.S. patent application Ser. No. 11/440,962, filed May 25, 2006, nowabandoned, which is a Divisional of U.S. patent application Ser. No.09/967,223, filed Sep. 28, 2001, now U.S. Pat. No. 7,052,686, issued May30, 2006, which claims benefit from U.S. Provisional Patent ApplicationNo. 60/236,596, filed Sep. 29, 2000, each of which is herebyincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to pegylated IL-10 and to methods for preparationof pegylated IL-10, and the like.

BACKGROUND OF THE INVENTION

The cytokine interleukin-10 (IL-10) is a dimer that becomes biologicallyinactive upon disruption of the non-covalent interactions connecting itstwo monomer subunits. IL-10 was first identified as a product of thetype 2 helper T cell and later shown to be produced by other cell typesincluding B cells and macrophages. It also inhibits the synthesis ofseveral cytokines produced from type 1 helper T cells, such asγ-interferon, IL-2, and tumor necrosis factor-α(TNF-α). The ability ofIL-10 to inhibit cell-mediated immune response modulators and suppressantigen-presenting cell-dependent T cell responses demonstrates IL-10has immunosuppressive properties. This cytokine also inhibitsmonocyte/macrophage production of other cytokines such as IL-1, IL-6,IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF),granulocyte colony-stimulating factor (G-CSF), and TNF-α. As a result ofits pleiotropic activity, IL-10 is under investigation for numerousclinical applications, such as for treating inflammatory conditions,bacterial sepsis, enterotoxin-induced lethal shock, and autoimmunediseases, e.g., rheumatoid arthritis, allograft rejection and diabetes.

IL-10 has a relatively short serum half-life in the body. For example,the half-life in mice as measured by in vitro bioassay or by efficacy inthe septic shock model system [see Smith et al., Cellular Immunology173:207-214 (1996)] is about 2 to 6 hours. A loss of IL-10 activity maybe due to several factors, including renal clearance, proteolyticdegradation and monomerization in the blood stream.

Pegylation of a protein can increase its serum half-life by retardingrenal clearance, since the PEG moiety adds considerable hydrodynamicradius to the protein. However, the conventional pegylationmethodologies are directed to monomeric proteins and larger, disulfidebonded complexes, e.g., monoclonal antibodies. Pegylation of IL-10presents problems not encountered with other pegylated proteins known inthe art, since the IL-10 dimer is held together by non-covalentinteractions. Dissociation of IL-10, which may be enhanced duringpegylation, will result in pegylated IL-10 monomers (PEG-IL-10monomers). The PEG-IL-10 monomers do not retain biological activity ofIL-10. It is also noted that di-PEG-IL-10, i.e., pegylation on two aminoacids residues of IL-10, does not retain significant in vitro biologicalactivity.

It would be an advantage to have an IL-10 product that is better able totolerate systemic exposure during treatment, by enhancing thecirculating life (delayed clearance), solubility and stability of IL-10,without disrupting the dimeric structure and affecting the activity ofIL-10. The present invention addresses this and other related needs inthe art.

SUMMARY OF THE INVENTION

This invention provides pegylated IL-10 (PEG-IL-10), more particularlymono-PEG-IL-10, methods for making the same, and pharmaceuticalcompositions containing mono-PEG-IL-10.

In one aspect, the invention is a mono-PEG-IL-10 which contains from oneto nine PEG molecules covalently attached via a linker to the alphaamino group of the amino acid residue at the N-terminus (amino terminus)of one subunit of the IL-10. Thus, this mono-PEG-IL-10 of the presentinvention can be expressed by the formula:

[X—O(CH₂CH₂O)_(n)]_(b)-L-NH-IL-10,

-   where L is a linker which comprises a C₂₋₁₂ alkyl;-   b represents from 1 to 9 PEG molecules covalently attached to the    linker L;-   n is from about 20 to 2300 representing the repeating units of each    PEG molecule attached to linker L, where n can be the same or    different for each PEG molecule, and the sum of the repeating units    represented by n for the PEG molecules does not exceed 2300;-   X is H or C₁₋₄ alkyl; and-   N is a nitrogen of the alpha amino group of the amino acid residue    at the N-terminus of one subunit of the IL-10 protein, which is    covalently attached to the linker L.-   Since the sum of n for all PEG molecules does not exceed 2300, the    total molecular mass of the PEG molecules attached to the linker    does not exceed about 100,000 Da.

In a specific embodiment, the linker L of a PEG-IL-10 contains a propylgroup (e.g., —CH₂CH₂CH₂—), which is attached at the amino terminus ofthe IL-10.

In another aspect, the invention provides pharmaceutical compositionscontaining stable mono-PEG-IL-10.

This invention further provides PEG-IL-10 compositions, where at least80% of the PEG-IL-10 is stable mono-PEG-IL-10. The present inventionalso provides PEG-IL-10 compositions, where the population ofmono-PEG-IL-10 is at least 80% positional isomer of mono-PEG-IL-10 whichis pegylated on the N-terminus of one subunit of IL-10.

In yet another aspect, this invention relates to a process for preparingPEG-IL-10, more particularly mono-PEG-IL-10, by reacting IL-10 with anactivated PEG-aldehyde linker in the presence of a reducing agent toproduce a PEG-IL-10. This process minimizes disruption of the dimericstructure of IL-10, such that there is little or no formation ofmonomeric proteins.

In a particular embodiment of a method of the present invention, aprocess for preparing a PEG-IL-10 includes reacting IL-10 with anactivated PEG-aldehyde linker in the presence of a sodiumcyanoborohydride where the molar ratio of IL-10 to sodiumcyanoborohydride is from about 1:5 to about 1:15 to form a PEG-IL-10 ata pH of about 6.3 to about 7.5 at a temperature of 18° C. to 25° C.,such that the linker is covalently attached to one amino acid residue ofthe IL-10.

These and other aspects of the invention are explained in greater detailin the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

All publications cited herein are expressly incorporated by reference intheir entireties.

This invention provides pegylated IL-10 (also referred to herein as“PEG-IL-10”). Preferably, a PEG-IL-10 of this invention contains one ormore polyethylene glycol (PEG) molecules covalently attached to only one(“mono”) amino acid residue of the IL-10 protein via a linker, such thatthe attachment of the PEG is stable. Thus, the pegylation occurs on asingle amino acid residue of IL-10 to provide mono-PEG-IL-10.

More than one PEG molecule can be attached to the single amino acidresidue via a linker that is capable of accommodating more than one PEGmolecule. A linker (for covalent attachment to IL-10) used in thisinvention preferably contains an aldehyde group, which chemically reactswith an amino or imino group of an amino acid residue, e.g., the alphaamino group at the N-terminus of the polypeptide, the epsilon aminogroup of lysine or an imino group of histidine. In order to obtain astable mono-PEG-IL-10 it is preferred that a PEG is attached at theamino terminus or to a lysine residue of IL-10 versus a histidineresidue. Most preferably, a mono-PEG-IL-10 of this invention containsone or more PEG molecules covalently attached via a linker to the alphaamino group of the N-terminus of only one subunit of IL-10. It is notedthat an IL-10 containing a PEG molecule is also known as a conjugatedprotein, whereas the protein lacking an attached PEG molecule can bereferred to as unconjugated.

Reaction conditions for pegylation are selected to minimize disruptionof the dimeric structure of IL-10. Thus, production of pegylated IL-10monomers, which lack IL-10 activity, is reduced by a method of theinvention, described infra. Preferably, the reaction conditions used ina method of the invention permit selective pegylation on the alpha aminogroup of the N-terminus of IL-10 (N-terminal-PEG-IL-10) to minimizeproduction of other PEG-IL-10 positional isomers, e.g., His-PEG-IL-10and Lys-PEG-IL-10. It is desirable, and advantageous, to have a singlepositional isomer of a therapeutic drug product for numerous reasons,including regulatory approval, consistent properties to allow betteranalytical characterization of the product in vivo and greaterconsistency and control of the process for making it.

A method of the present invention provides compositions containing apopulation of stable mono-PEG-IL-10, i.e., the PEG moiety of amono-PEG-IL-10 is not hydrolyzed from the pegylated amino acid residueusing a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH 6.5) over 8to 16 hours at room temperature. Thus, populations of stablemono-PEG-IL-10 (described supra) can be achieved in PEG-IL-10compositions, where greater than 80% of the composition is stablemono-PEG-IL-10, more preferably at least 90%, and most preferably atleast 95%. However, greater than 98% mono-PEG-IL-10 can be achieved in aPEG-IL-10 composition, as shown in the Example infra. Furthermore, amethod of the present invention provides PEG-IL-10 compositionscontaining a substantially homogeneous population of mono-PEG-IL-10,where at least 80%, more preferably at least 90%, of the mono-PEG-IL-10is a positional isomer which is pegylated at the N-terminus of IL-10.

Interleukin-10

General methods of molecular biology are described in, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2d ed. (1989); and Brent, etal. Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork, Ausubel et al., eds. (1988 and periodic supplements).

An “interleukin-10” or “IL-10” protein used in this invention, whetherconjugated to a polyethylene glycol, i.e., PEG-IL-10, or in anon-conjugated form, is a protein comprising two subunits (monomers)joined by non-covalent interactions to form a dimer. The terms“monomeric IL-10” and “IL-10 monomer” refer to one subunit of IL-10,which does not possess biological activity of native IL-10. Thus, anIL-10 used in this invention to make PEG-IL-10 is a dimer whichpossesses activity of native IL-10.

An IL-10 protein used in the present invention contains an amino acidsequence that shares an observed homology of at least 75%, morepreferably at least 85%, and most preferably at least 90% or more, e.g.,at least 95%, with the sequence of a mature IL-10 protein, i.e., lackingany leader sequences. See, e.g., U.S. Pat. No. 6,217,857. Amino acidsequence homology, or sequence identity, is determined by optimizingresidue matches and, if necessary, by introducing gaps as required.Homologous amino acid sequences are typically intended to includenatural allelic, polymorphic and interspecies variations in eachrespective sequence. Typical homologous proteins or peptides will havefrom 25-100% homology (if gaps can be introduced) to 50-100% homology(if conservative substitutions are included) with the amino acidsequence of the IL-10 polypeptide. See Needleham et al., J. Mol. Biol.48:443-453 (1970); Sankoff et al. in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison, 1983,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif., and the University of WisconsinGenetics Computer Group, Madison, Wis.

The IL-10 can be glycosylated or unglycosylated. Muteins or otheranalogs, including the BCRF1 (Epstein Barr Virus viral IL-10) protein,can also be used. Modifications of sequences encoding IL-10 can be madeusing a variety of techniques, e.g., site-directed mutagenesis [Gillmanet al., Gene 8:81-97 (1979); Roberts et al., Nature 328:731-734 (1987)],and can be evaluated by routine screening in a suitable assay for IL-10activity. Modified IL-10 proteins, e.g., variants, can vary from thenaturally-occurring sequence at the primary structure level. Suchmodifications can be made by amino acid insertions, substitutions,deletions and fusions. IL-10 variants can be prepared with variousobjectives in mind, including increasing serum half-life, reducing animmune response against the IL-10, facilitating purification orpreparation, decreasing conversion of IL-10 into its monomeric subunits,improving therapeutic efficacy, and lessening the severity or occurrenceof side effects during therapeutic use. The amino acid sequence variantsare usually predetermined variants not found in nature, although othersmay be post-translational variants, e.g., glycosylated variants. Anyvariant of IL-10 can be used in this invention provided it retains asuitable level of IL-10 activity.

IL-10 used in this invention can be derived from a mammal, e.g. human ormouse. Human IL-10 (hIL-10) is preferred for treatment of humans in needof IL-10 treatment. IL-10 used in this invention is preferably arecombinant IL-10.

Methods describing the preparation of human and mouse IL-10 can be foundin U.S. Pat. No. 5,231,012. In another embodiment of the presentinvention, IL-10 can be of viral origin. The cloning and expression of aviral IL-10 from Epstein Barr virus (BCRF1 protein) is disclosed inMoore et al., Science 248:1230 (1990).

IL-10 can be obtained in a number of ways using standard techniquesknown in the art, e.g., isolated and purified from culture media ofactivated cells capable of secreting the protein (e.g., T-cells),chemically synthesized, or recombinant techniques, (see, e.g.,Merrifield, Science 233:341-47 (1986); Atherton et al., Solid PhasePeptide Synthesis, A Practical Approach, 1989, I.R.L. Press, Oxford;U.S. Pat. No. 5,231,012 which teaches methods for the production ofproteins having IL-10 activity, including recombinant and othersynthetic techniques). Preferably, IL-10 protein is obtained fromnucleic acids encoding the IL-10 polypeptide using recombinanttechniques. Recombinant human IL-10 is also commercially available,e.g., from PeproTech, Inc., Rocky Hill, N.J.

IL-10 exhibits several biological activities, which could form the basisof assays and units. See, e.g., Current Protocols in Immunology, JohnWiley & Sons, NY Coligan et al., eds., (1991 and periodic supplements).IL-10 activity is described in, e.g., U.S. Pat. No. 5,231,012 and inInternational Patent Publication No. WO 97/42324, which provide in vitroassays suitable for measuring such activity. In particular, IL-10inhibits the synthesis of at least one cytokine in the group consistingof IFN-γ, lymphotoxin, IL-2, IL-3, and GM-CSF in a population of Thelper cells induced to synthesize one or more of these cytokines byexposure to antigen and antigen presenting cells (APCs). IL-10 also hasthe property of stimulating cell growth, and by measuring cellproliferation after exposure to the cytokine, IL-10 activity can bedetermined.

As already described above, the activity of a mono-PEG-IL-10 of thepresent invention can be determined using a standard IL-10 activityassay known in the art. Preferably, mono-PEG-IL-10 retains at least 5%activity of the unconjugated IL-10. Activity greater than 30% isattainable from a mono-PEG-IL-10 of this invention, as demonstrated inExample 1. Preferably, a mono-PEG-IL-10 of the invention hassignificantly increased bioavailability in the body of a patientcompared with the unconjugated IL-10, e.g., as shown by Example 2.

Polyethylene Glycol

It shall be appreciated by those having ordinary skill in the art thatvarious polymers can be used in addition to PEG for attachment at theN-terminus of one monomer of IL-10, such as polyoxyethylene2-methyl-2-propenyl methyl diether, or polyoxyethyleneallylmethyldiether, however PEG is most preferred. Thus, by way ofexample, PEG is used to describe this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented by the formula:

X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH,  (1)

where n is 20 to 2300 and X is H or a terminal modification, e.g., aC₁₋₄ alkyl.

Preferably, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). It is notedthat the other end of the PEG, which is shown in formula (I) terminatingin OH, covalently attaches to a linker moiety via an ether oxygen bond.

When used in a chemical structure, the term “PEG” includes the formula(I) above without the hydrogen of the hydroxyl group shown, leaving theoxygen available to react with a free carbon atom of a linker of theinvention to form an ether bond.

Any molecular mass for a PEG can be used as practically desired, e.g.,from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300). Thenumber of repeating units “n” in the PEG is approximated for themolecular mass described in Daltons. It is preferred that the combinedmolecular mass of PEG on an activated linker is suitable forpharmaceutical use. Thus, the combined molecular mass of the PEGmolecules should not exceed 100,000 Da. For example, if three PEGmolecules are attached to a linker, where each PEG molecule has the samemolecular mass of 12,000 Da (each n is about 270), then the totalmolecular mass of PEG on the linker is about 36,000 Da (total n is about820). The molecular masses of the PEG attached to the linker can also bedifferent, e.g., of three molecules on a linker two PEG molecules can be5,000 Da each (each n is about 110) and one PEG molecule can be 12,000Da (n is about 270).

Preferably, the combined or total molecular mass of PEG used in aPEG-IL-10 is from about 3,000 Da to 60,000 Da (total n is from 70 to1,400), more preferably from about 10,000 Da to 36,000 Da (total n isabout 230 to about 820). The most preferred combined mass for PEG isfrom about 12,000 Da to 24,000 Da (total n is about 270 to about 550).

One skilled in the art can select a suitable molecular mass for the PEG,e.g., based on how the pegylated IL-10 will be used therapeutically, thedesired dosage, circulation time, resistance to proteolysis,immunogenicity, and other considerations. For a discussion of PEG andits use to enhance the properties of proteins, see N. V. Katre, AdvancedDrug Delivery Reviews 10: 91-114 (1993).

Activated PEG

To conjugate PEG to IL-10, an activated linker covalently attached toone or more PEG molecules is reacted with an amino or imino group of anamino acid residue, most preferably with an alpha amino group at theN-terminus of IL-10, to form a mono-PEG-IL-10 of the present invention.

A linker is “activated” if it is chemically reactive and ready forcovalent attachment to an amino group on an amino acid residue. Anyactivated linker can be used in this invention provided it canaccommodate one or more PEG molecules and form a covalent bond with anamino group of an amino acid residue under suitable reaction conditions.Preferably, the activated linker attaches to an alpha amino group in ahighly selective manner over other attachment sites, e.g., epsilon aminogroup of lysine or imino group of histidine.

Activated PEG can be represented by the formula:

(PEG)_(b)-L′,  (2)

where PEG (defined supra) covalently attaches to a carbon atom of thelinker to form an ether bond, b is 1 to 9 (i.e. 1 to 9 PEG molecules canbe attached to the linker), and L′ contains a reactive group (anactivated moiety) which can react with an amino or imino group on anamino acid residue to provide a covalent attachment of the PEG to IL-10.

A preferred activated linker (L′) of the invention contains an aldehydeof the formula RCHO, where R is a linear (straight chain) or branchedC₁₋₁₁ alkyl. After covalent attachment of an activated linker to IL-10,the linker (referred to as “-L-” in the structural formulas recitedherein) between the IL-10 and PEG contains 2 to 12 carbon atoms.

Propionaldehyde is an example of a preferred activated linker of thisinvention. PEG-propionaldehyde, represented in formula (3), is describedin U.S. Pat. No. 5,252,714 and is commercially available from ShearwaterPolymers (Huntsville, Ala.).

PEG-CH₂CH₂CHO  (3)

If it is desirable to covalently attach more than one PEG molecule toIL-10, then a suitable activated branched (also known as “multi-armed”)linker can be used. Any suitable branched PEG linker that covalentlyattaches two or more PEG molecules to an amino group on an amino acidresidue of IL-10, more preferably to an alpha amino group at theN-terminus, can be used. Preferably, a branched linker used in thisinvention contains two or three PEG molecules.

For example, a branched PEG linker used in this invention can be alinear or branched aliphatic group that is hydrolytically stable andcontains an activated moiety, e.g., an aldehyde group, which reacts withan amino group of an amino acid residue, as described above. Preferably,the aliphatic group of a branched linker contains 2 to 12 carbons. Forexample, an aliphatic group can be a t-butyl which contains as many asthree PEG molecules on each of three carbon atoms (i.e., a total of 9PEG molecules) and a reactive aldehyde moiety on the fourth carbon ofthe t-butyl.

Examples of activated, branched PEG linkers are also described in U.S.Pat. Nos. 5,643,575, 5,919,455, and 5,932,462. One having ordinary skillin the art can prepare modifications to branched PEG linkers as desired,e.g., addition of a reactive aldehyde moiety.

Methods for the preparation of linkers for use in the present inventionare well known in the art, e.g., see U.S. Pat. Nos. 5,643,575,5,919,455, and 5,932,462. Activated PEG-linkers, such as PEG-aldehydes,can be obtained from a commercial source, e.g., Shearwater Polymers,(Huntsville, Ala.) or Enzon, Inc. (Piscataway, N.J.).

Pegylated IL-10

A mono-PEG-IL-10 of this invention is an IL-10 that has a linkercontaining one or more PEG molecules, which is covalently attached toonly one amino acid residue of the IL-10. Preferred mono-PEG-IL-10molecules of the invention contain a PEG-linker attached to an aminoacid residue of IL-10 to form a hydrolytically stable bond, e.g., on thealpha amino group at the N-terminus or on the side chain of a lysineresidue. (The stability of a mono-PEG-IL-10 protein of the invention canbe determined by a conventional hydroxylamine assay, e.g., usingconditions as described above.) Most preferably, the PEG is attached atthe N-terminus of an IL-10 subunit on the nitrogen atom of the alphaamino group. Thus, over an entire IL-10 protein containing two subunits,only one subunit is pegylated on one amino acid residue.

A preferred mono-PEG-IL-10 of the invention is represented by thestructural formula:

(PEG)_(b)-L-NH-IL-10  (4)

where PEG, b, and L (linker) are as described, and N is nitrogen of anamino or imino group of an amino acid residue on one subunit of IL-10.If b is greater than one, then L must be a suitable linker whichattaches two or more PEG molecules to an amino acid residue of IL-10.

For example, if the linker is a PEG-propionaldehyde and b is 1 then uponcovalent attachment of the linker to an amino acid residue of the IL-10,the PEG-IL-10 can have a structural formula (5) as shown:

PEG-CH₂CH₂CH₂—NH-IL-10.  (5)

Conjugation Reaction Between Peg and IL-10

Although not intending to limit the scope of the invention to any onetheory, the following schematic illustrates a reaction between anactivated PEG aldehyde linker and an amino or imino group of an aminoacid residue of one of the IL-10 monomers:

PEG-R—CHO+NH₂-IL-10← - - - →PEG-R—CH═N-IL-10  (6)

where R is a C₁₋₁₁ alkyl and N is nitrogen of a reactive amino group onan amino acid residue of IL-10. In reaction (6), the activated PEGcovalently attaches to the IL-10 to form an imine linkage. Reduction ofthe imine linkage by the reducing agent, e.g., sodium cyanoborohydride(Sigma-Aldrich, St. Louis, Mo.) forms pegylated IL-10, as shown:

PEG-R—CH═N-IL-10+NaCNBH₃→PEG-R—CH₂—NH-IL-10.  (7)

Other reducing agents can be used instead of sodium cyanoborohydride inthis reaction, including sodium borohydride, tertiary butyl amineborane, sodium triacetyl borohydide, dimethylamine borate,trimethylamine borate, and pyridine borate. Sodium cyanoborohydride ispreferred because it specifically reduces an imine linkage, which isformed between an aldehyde group of the activated PEG and amino group ofthe amino acid of IL-10.

As shown in reactions (6) and (7), a Schiff base is formed during thepreparation of mono-PEG-IL-10. There was concern that this intermediate,which is very difficult to separate from mono-PEG-IL-10, could lower thepurity of the mono-PEG-IL-10 if the intermediate accumulates in thereaction and is not reduced to the product. Typically, this problem isavoided by using higher concentrations of reducing agent, e.g., seeKinstler et al., Pharm. Res. 13:996-1002 (1996) and Chamow et al.,Bioconjugate Chem. 5: 133-140 (1994). However, there was further concernthat the higher concentrations of reducing agent, e.g., sodiumcyanoborohydride, used conventionally would disrupt the dimericstructure of IL-10. For example, as little as about 14 mM sodiumcyanoborohydride can result in more than 10% monomerization of IL-10.

It was discovered during the development of a method of the presentinvention that reaction (6) is reversible and that equilibrium greatlyfavors the hydrolysis of the Schiff base (i.e., the imine) reactionintermediate. It is believed that the intermediate is very unstable andis quickly hydrolyzed in the absence of reducing agent. This wasdemonstrated by incubating activated PEG linker with IL-10 in theabsence of a reducing agent. No pegylated IL-10 (unreduced or reduced)was detectable by size exclusion (SE)-HPLC after 24 hours, however theaddition of sodium cyanoborohydride produced mono-PEG-IL-10 of theinvention instantly. Based on these results, much lower concentrationsof a reducing agent are used to prepare PEG-IL-10 of the presentinvention in comparison with concentrations of reducing agent taught inthe art. This is an advantage over conventional methods of pegylatingproteins because although a low concentration of reducing agent isemployed the purity of the mono-IL-10 product is not reduced, i.e., nointermediate is present, and IL-10 monomerization is controlled.

The ratio of IL-10 to the reducing agent can be from about 1:0.5 to1:50, more preferably from about 1:1 to 1:30. Most preferably, the molarratio of IL-10 to the reducing agent is from about 1:5 to 1:15 tominimize any disruption of the IL-10 during pegylation. Incubating molarratios of less than 10:1 of sodium cyanoborohydride to IL-10 in a methodof the present invention does not disrupt the dimeric structure ofIL-10. The ability to use lesser amounts of reducing agent to reduce theSchiff base into a secondary amine, thereby accomplishing pegylation,was surprising in light of the teachings in the art, i.e., significantlyhigher molar ratios of sodium cyanoborohydride to protein is necessaryto pegylate proteins (e.g., about 75:1 to 350:1; see, e.g., Kinstler etal., supra and Chamow et al., supra).

In a method of the present invention, the molar ratio of the activatedPEG linker to IL-10 can be from about 0.5:1 to 20:1, more preferably 2:1to 8:1.

Various aqueous buffers can be used in the present method to catalyzethe covalent addition of PEG to IL-10. The pH of a buffer used is fromabout 5.5 to 7.8, more preferably the pH is in a neutral range, e.g.,from about 6.3 to 7.5. In order not to disrupt the non-covalentinteractions between the two subunits of IL-10, it is desirable tomaintain IL-10 in this neutral pH range, in particular, during thepegylation reaction. This neutral pH range also increases thesite-specific pegylation of IL-10 at the alpha amino group of theN-terminus versus other imino or amino groups of other amino acidresidues, e.g., lysine or histidine. Buffers having a pKa close toneutral pH range are preferred, e.g., phosphate buffer. Preferably,buffers and pH are selected that do not result in monomerization ofIL-10. IL-10 monomerization can be detected and monitored usingconventional SE-HPLC.

The temperature range for preparing a mono-PEG-IL-10 of the invention isfrom about 5° C. to 30° C. More preferably, the temperature is fromabout 18° C. to 25° C.

The pegylation reaction can proceed from 3 to 48 hours, more preferably10 to 24 hours. The reaction can be monitored using SE-HPLC, which candistinguish IL-10, mono-PEG-IL-10 and di-PEG-IL-10 (i.e., pegylationoccurs on two amino acid residues of IL-10, typically on both subunits).It is noted that mono-PEG-IL-10 forms before di-PEG-IL-10. When themono-PEG-IL-10 concentration reaches a plateau, the reaction can beterminated by adding glycine solution to quench any remaining activatedPEG. Using reaction conditions according to a method of the invention,typically 5 to 10% di-PEG-IL-10 and 38% to 43% mono-PEG-IL-10 is formed(the remainder being unmodified IL-10).

Conventional separation and purification techniques known in the art canbe used to purify mono-PEG-IL-10, such as size exclusion (e.g. gelfiltration) and ion exchange chromatography, which can separatepegylated IL-10 monomers and di-PEG-IL-10 from the mono-PEG-IL-10 of theinvention.

It may be desirable to polish or resolve a population of mono-PEG-IL-10in a PEG-IL-10 composition prepared according to a method of the presentinvention. The polishing step separates less stable mono-PEG-IL-10 (e.g.His-PEG-IL-10) from stable mono-PEG-IL-10 (e.g. N-terminus-PEG-IL-10 orLys-PEG-IL-10), and thus can achieve greater homogeneity of stablepositional isomers, e.g., greater than 95% of a PEG-IL-10 composition.Less stable positional isomers of PEG-IL-10, e.g., histidine-PEG-IL-10,can be hydrolyzed during a polishing step. The population of PEG-IL-10can be incubated in an aqueous buffer, preferably a TRIS buffer (e.g.,10 to 300 mM, more preferably about 30 to 70 mM), at about pH 5.0 to9.0, more preferably pH 7.0 to 8.0 at 15° C. to 35° C. overnight.Alternatively, the population of PEG-IL-10 can be treated with 0.05 to0.4 M hydroxylamine HCl salt (pH about 6.5) at room temperature for 0.5to 10 hours. Hydrolyzed IL-10 and PEG remnant can be removed from thepopulation of stable mono-PEG-IL-10 by a separation/purification stepusing, e.g., gel filtration or ion exchange chromatography.

In U.S. Pat. No. 5,985,265 the pegylation of interferon using analdehyde linker was accomplished at acidic pH 4.0 at 4° C. Pegylation ofIL-10 at this pH would result in its monomerization and loss ofbiological activity. Conventional wisdom in the art teaches that formost activated PEG, as the reaction pH is increased under basicconditions pegylation occurs at more stable sites on the protein. Forexample, succinimidyl carbonate-PEG forms about 90% Lys-PEG-IL-10 (morestable) and about 10% His-PEG-IL-10 (less stable) at a reaction pH 8.8,and about 64% Lys-PEG-IL-10 and 36% His-PEG-IL-10 at a reaction pH 6.3.However, during the discovery of the present invention, it wasdetermined that as the pH of a pegylation reaction using an aldehydelinker is increased above a neutral pH range, pegylation occurs morefrequently at less stable sites of IL-10, e.g., His-PEG-IL-10, therebyforming a heterogenous mixture of unstable PEG-IL-10. Thus, it wasunexpected that IL-10 pegylation using an aldehyde linker in neutral pHrange would provide a highly stable and homogeneous population ofpegylated IL-10.

Pharmaceutical Compositions Containing Peg-Il-10

A PEG-IL-10 of this invention is useful in the treatment of conditionswhich are treatable with IL-10, e.g., diseases associated with undesiredT-cell activation and T-cell expansion such as autoimmune diseases,organ and bone marrow transplant rejection, graft-versus-host disease,parasitic infections, granulomas, inflammatory diseases, Crohn'sdisease, colitis, pancreatitis, inflammatory lung, eye diseases,allergic conditions, asthma, atopic dermatitis, and rhinitis.

PEG-IL-10 can be formulated in a pharmaceutical composition comprising atherapeutically effective amount of the IL-10 and a pharmaceuticalcarrier. A “therapeutically effective amount” is an amount sufficient toprovide the desired therapeutic result. Preferably, such amount hasminimal negative side effects. The amount of PEG-IL-10 administered totreat a condition treatable with IL-10 is based on IL-10 activity of theconjugated protein, which can be determined by IL-10 activity assaysknown in the art. The therapeutically effective amount for a particularpatient in need of such treatment can be determined by consideringvarious factors, such as the condition treated, the overall health ofthe patient, method of administration, the severity of side-effects, andthe like.

The therapeutically effective amount of pegylated IL-10 can range fromabout 0.01 to about 100 μg protein per kg of body weight per day.Preferably, the amount of pegylated IL-10 ranges from about 0.1 to 20 μgprotein per kg of body weight per day, more preferably from about 0.5 to10 μg protein per kg of body weight per day, and most preferably fromabout 1 to 4 μg protein per kg of body weight per day. Less frequentadministration schedules can be employed using the PEG-IL-10 of theinvention since this conjugated form is longer acting than IL-10. Thepegylated IL-10 is formulated in purified form and substantially free ofaggregates and other proteins. Preferably, IL-10 is administered bycontinuous infusion so that an amount in the range of about 50 to 800 μgprotein is delivered per day (i.e., about 1 to 16 μg protein per kg ofbody weight per day PEG-IL-10). The daily infusion rate may be variedbased on monitoring of side effects and blood cell counts.

To prepare pharmaceutical compositions containing mono-PEG-IL-10, atherapeutically effective amount of PEG-IL-10 is admixed with apharmaceutically acceptable carrier or excipient. Preferably the carrieror excipient is inert. A pharmaceutical carrier can be any compatible,non-toxic substance suitable for delivering the IL-10 compositions ofthe invention to a patient. Examples of suitable carriers include normalsaline, Ringer's solution, dextrose solution, and Hank's solution.Non-aqueous carriers such as fixed oils and ethyl oleate may also beused. Nonaqueous carriers such as fixed oils and ethyl oleate may alsobe used. A preferred carrier is 5% dextrose/saline. The carrier maycontain minor amounts of additives such as substances that enhanceisotonicity and chemical stability, e.g., buffers and preservatives.

Compositions of the invention can be administered orally or injectedinto the body. Formulations for oral use can also include compounds tofurther protect the IL-10 from proteases in the gastrointestinal tract.Injections are usually intramuscular, subcutaneous, intradermal orintravenous. Alternatively, intra-articular injection or other routescould be used in appropriate circumstances. When administeredparenterally, pegylated IL-10 is preferably formulated in a unit dosageinjectable form (solution, suspension, emulsion) in association with apharmaceutical carrier. See, e.g., Avis et al., eds., PharmaceuticalDosage Forms: Parenteral Medications, Dekker, N.Y. (1993); Lieberman etal., eds., Pharmaceutical Dosage Forms: Tablets, Dekker, N.Y. (1990);and Lieberman et al., eds., Pharmaceutical Dosage Forms: DisperseSystems, Dekker, N.Y. (1990). Alternatively, compositions of theinvention may be introduced into a patient's body by implantable orinjectable drug delivery system, e.g., Urquhart et al. Ann. Rev.Pharmacol. Toxicol. 24:199-236, (1984); Lewis, ed., Controlled Releaseof Pesticides and Pharmaceuticals Plenum Press, New York (1981); U.S.Pat. No. 3,773,919; U.S. Pat. No. 3,270,960; and the like. The pegylatedIL-10 can be administered in aqueous vehicles such as water, saline orbuffered vehicles with or without various additives and/or dilutingagents.

Preparation of Such Pharmaceutical Compositions is Known in the Art;see, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia:National Formulary, Mack Publishing Company, Easton, Pa. (1984).

Example 1

In this Example human IL-10 (hIL-10) was used, however, other forms ofIL-10, e.g., mouse, viral or an IL-10 variant, can be used withoutaffecting the pegylation reaction.

The conjugation reaction was performed at pH 6.3 in an attempt tomaximize the probability of site-specific pegylation at the N-terminusof one subunit of IL-10 without disrupting the IL-10 structure. Themolar ratio of IL-10 to reducing agent (sodium cyanoborohydride;Sigma-Aldrich, St. Louis, Mo.) was 1:4.5, which is much less than themolar ratios of protein to reducing agent taught in the art, e.g., 1:75to 1:350 (see Kinstler et al., supra and Chamow et al., supra). Sizeexclusion-HPLC showed less than 1% of the IL-10 was monomeric by the endof the reaction.

In an attempt to determine the effect of reducing agent concentration onIL-10 stability, i.e., IL-10 dissociation into its subunits, 0.1 mMIL-10 was incubated with varying concentrations of sodiumcyanoborohydride (none, 0.5, 1.0, 2.5, 5.0 and 14 mM) at pH 6.3 for 15hours to determine whether monomerization of IL-10 is a function of theratio of protein to reducing agent (1:5, 1:10, 1:25, 1:50 and 1:140).The results showed that at higher ratios of reducing agent to proteinthere were greater levels of IL-10 monomer formed. Surprisingly, at amolar ratio of less than 1:10 of IL-10 to reducing agent, disruption ofthe dimeric structure of hIL-10 was negligible.

As a result of this discovery, the pegylation reaction was modifiedaccordingly. Purified hIL-10 was dialyzed against different reactionbuffers at pH 6.3 and 8.6 (50 mM sodium phosphate, 100 mM sodiumchloride, pH 6.3; or 50 mM sodium phosphate, 10 mM sodiumtetraborohydrate, 100 mM sodium chloride, pH 8.6). The IL-10 was dilutedto 4 mg/ml (0.1 mM in each buffer). Two activated PEG molecules,methoxy-PEG-aldehyde MW 5000 and MW 12000 (Shearwater), were added toeach reaction buffer in approximately a 1:1 molar ratio of IL-10 to PEG(in separate studies). Aqueous sodium cyanoborohydride was added to thereaction mixture to a final concentration of 0.5 to 0.75 mM (about 1:4.5to 1:6.8 IL-10 to reducing agent). The pegylation reaction was carriedout at room temperature (18 to 25° C.) for 15 to 20 hours until thedesired degree of mono-pegylation, i.e., covalent addition of PEG to onesubunit of IL-10, was achieved. The reaction was quenched with glycine.The final reaction solution was analyzed by SE-HPLC to determine thepercentage of mono-PEG-IL-10 based on concentrations of hIL-10,mono-PEG-IL-10 and di-PEG-IL-10 and by reverse phase (rp)-HPLC todetermine levels of positional isomers. The mono-PEG-IL-10 was thenpurified from unreacted hIL-10, activated PEG-linker and di-PEG-IL-10 bygel filtration chromatography and characterized by rp-HPLC and bioassay(e.g., stimulation of proliferation of BaMR29α1 cells, which is a murineB-cell line created by transfecting Ba/F3 cells with the murine IL-10receptor cDNA).

Three purified (SE-HPLC) mono-PEG-IL-10 samples contained greater than98% mono-PEG-IL-10. Contrary to expectations (i.e. pegylation rates ofproteins generally occur at higher pH to form a more stable pegylatedprotein), the rate of formation of stable mono-PEG-IL-10 (wherepegylation occurs at the alpha amino group or on lysine) was higher atpH 6.3 than at pH 8.6. Most importantly, SE-HPLC showed no increase inIL-10 monomer at either pH.

Reverse phase HPLC analysis of the final reaction mixtures showed mainlya single positional isomer demonstrating increased selectivity forN-terminal pegylation at pH 6.3, while multiple peaks representingdifferent positional isomers of mono-PEG-IL-10 were observed underhigher pH (8.6) conditions. N-terminal amino acid sequencing of themono-PEG-IL-10 purified from the pH 6.3 reaction mixture indicated thatover 40% of PEG-IL-10 was N-terminally blocked. Unlike the hydroxylamineassay, amino acid sequencing measures blockage per monomer. The maximumpossible blockage for N-terminally pegylated dimers that are pegylatedat a stoichiometry of one PEG-linker per dimer is 50%. Therefore, lessthan 20% of this preparation was pegylated at sites other than theN-terminus of IL-10 according to sequence analysis, i.e., greater than80% pegylation at the N-terminus. However, according to rp-HPLC at least90% of the IL-10 was pegylated at the N-terminus. Moreover, at least 95%of the pegylated IL-10 was stable according to the hydroxylamine assay.The purified mono-PEG-IL-10 was also biologically active, demonstratingabout 32% of the specific biological activity of unmodified hIL-10.

Thus, IL-10 was successfully pegylated at the amino terminus using twoPEG-aldehyde linker molecules having different molecular weight PEGmolecules in a site-specific manner using concentrations of reducingagent below conventional levels. Structurally intact mono-PEG-IL-10 canbe formed in high yield as a homogenous population, i.e., asubstantially single positional isomer, using this method.

Example 2

Previous studies showed the ability of recombinant human IL-10 tosuppress the production of pro-inflammatory cytokines in LPS-primed mice(with C. parvum) given a lethal dose of lipopolysaccharide (LPS). Theprimed mice produce high levels of TNF-α and IFN-γ, which are majorcytokine mediators of LPS lethality. Recombinant human IL-10 was mosteffective in suppressing the production of cytokines when administeredto C. parvum mice simultaneously or one hour at most prior to LPSexposure.

In this Example the C. parvum-mouse model was used to compare theduration and extent of suppressive effect of two mono-PEG-IL-10 proteinson cytokine responses triggered by LPS. It demonstrates that amono-PEG-IL-10 of the present invention maintains biological activity invivo and has a reduced serum clearance compared with unmodified IL-10.

BDF-1 mice were challenged with LPS, one week after priming with C.parvum, according to the method of Smith et al., supra. Mice were bled1.5 hours after the challenge to determine the levels of circulatingTNF-α and 3 hours after challenge to measure circulating levels ofIL-12, IL-6 and IFN-γ. IFN-γ and IL-12 responses were inhibited to thesame extent by IL-10, and thus IFN-γ data is not shown.

Two different PEG propionaldehydes (PALD) which contain 12,000 or 20,000Da PEG were used in this study. The mono-PEG-IL-10 proteins wereadministered subcutaneously (8×10⁵ units) to mice 20, 48 or 72 hoursbefore the LPS challenge, and also administered to mice simultaneouslywith LPS to show an initial level of IL-10 activity that could befollowed over time. The amount of protein administered to the mice wasequalized for each pegylated IL-10 based on specific activity, which wasdetermined using an in vitro bioassay. Control mice were given mouseserum albumin as an inert protein preparation.

The data in the Table below show that PEG-IL-10 inhibits expression ofpro-inflammatory cytokines in vivo when administered 48 hours (forPEG-12K) and 72 hours (for PEG-20K) before the LPS challenge. Incontrast, native IL-10 was efficacious only when co-administered withLPS. This is likely due to a serum half-life of less than 5 hours fornative IL-10, as shown previously in pharmacokinetic (PK) studies. Thedata from this Example demonstrate that mono-PEG-IL-10 does not lead torapid monomerization in vivo, and is thus maintained in the body, i.e.,without renal clearance.

TABLE IL-10 preparation 0 hr -20 hr -48 hr -72 hr TNF-α (% inhibition)Non-pegylated IL-10 (10 μg) 62 7 8 0 PALD-12K (34 μg) 69 93 68 17PALD-20K (73 μg) 61 85 96 78 IL-12p40 (% inhibition) Non-pegylated IL-10(10 μg) 88 2 0 8 PALD-12K (34 μg) 87 94 53 20 PALD-20K (73 μg) 84 92 9163 IL-6 (% inhibition) Non-pegylated IL-10 (10 μg) 44 15 9 15 PALD-12K(34 μg) 58 73 73 15 PALD-20K (73 μg) 29 91 91 82

The preceding Examples demonstrate experiments performed to furtherteach the invention. It shall be appreciated by those skilled in the artthat the particular embodiments disclosed in the Examples are forpurposes of illustration. Numerous equivalent variations of the methodsand compositions described can be employed to achieve a substantiallysimilar result without departing from the spirit of the invention

1. A mono-PEG-IL-10.
 2. The mono-PEG-IL-10 of claim 1, comprising one ortwo PEG molecules covalently attached via a linker to one amino acidresidue on IL-10, wherein the attachment is at an N-terminal amino acidresidue or on a lysine residue.
 3. The mono-PEG-IL-10 of claim 2: (a)which comprises a methoxy PEG; (b) wherein the IL-10 is human IL-10; (c)wherein the total molecular mass of all PEG covalently attached to thelinker is from 3,000 daltons to 60,000 daltons; or (d) wherein thelinker is a linear or branched C₁₋₁₁ alkyl.
 4. The mono-PEG-IL-10 ofclaim 2, wherein the total molecular mass of all PEG covalently attachedto the linker is from 10,000 daltons to 36,000 daltons.
 5. Themono-PEG-IL-10 of claim 2, wherein the linker is a linear C₃ alkyl. 6.The mono-PEG-IL-10 of claim 1, wherein a PEG molecule is covalentlyattached to the alpha amino group of one N-terminus of IL-10 via alinear C₃ alkyl linker.
 7. A PEG-IL-10 comprising the formula:[X—O(CH₂CH₂O)_(n)]_(b)-L-NH-IL-10, where X is H or C₁₋₄ alkyl, n is 20to 2300, b is 1 to 9 and L is a C₁₋₁₁ alkyl linker moiety which iscovalently attached to nitrogen (N) of the alpha amino group at theamino terminus of one IL-10 subunit, provided that when b is greaterthan 1 the total of n does not exceed
 2300. 8. A PEG-IL-10 of claim 7,wherein L is —CH₂CH₂CH₂—.
 9. A pharmaceutical composition, comprising amono-PEG-IL-10 of claim 1 in combination with a pharmaceuticallyacceptable carrier.
 10. A method of treating inflammation in anindividual in need of such treatment, comprising administering to theindividual a therapeutically effective amount of a pharmaceuticalcomposition of claim
 9. 11. A process for preparing a mono-PEG-IL-10,comprising the step of: reacting IL-10 with an activated PEG-aldehydelinker in the presence of a reducing agent to form the mono-PEG-IL-10,wherein the linker is covalently attached to one amino acid residue ofthe IL-10.
 12. The process of claim 11 wherein: (a) the reducing agentis sodium cyanoborohydride; (b) the activated PEG-aldehyde linker isPEG-propionaldehyde; (c) the PEG is a methoxy-PEG; (d) the linker ismulti-armed; (e) the ratio of IL-10 to the sodium cyanoborohydride isfrom about 1:0.5 to 1:50; (f) the total molecular mass of all PEGcomprising the PEG-aldehyde linker is from 3,000 daltons to 60,000daltons; or (g) the reacting step is performed at a pH of 5.5 to 7.8.13. The process of claim 11, wherein the ratio of IL-10 to the sodiumcyanoborohydride is 1:5 to 1:15.
 14. The process of claim 11, whereinthe total molecular mass of all PEG comprising the PEG-aldehyde linkeris from 10,000 daltons to 36,000 daltons.
 15. The process of claim 11,wherein the reacting step is performed at a pH of 6.3 to 7.5.
 16. Theprocess of claim 11, further comprising a step selected from: incubatingthe mono-PEG-IL-10 product in a buffer at pH 5.0 to 9.0; and treatingthe mono-PEG-IL-10 product with 0.05 to 0.4 M hydroxylamine HCl salt.17. A process for preparing a mono-PEG-IL-10, comprising the step of:reacting IL-10 with an activated PEG-propionaldehyde linker in thepresence of sodium cyanoborohydride, wherein the molar ratio of IL-10 tosodium cyanoborohydride is from about 1:5 to about 1:15, at a pH ofabout 6.3 to about 7.5 and a temperature of from 18° C. to 25° C. toform the mono-PEG-IL-10, wherein the linker is covalently attached toone amino acid residue of the IL-10.
 18. The process of claim 17,wherein the total molecular mass of all PEG comprising the PEG-aldehydelinker is from 10,000 daltons to 36,000 daltons.
 19. The process ofclaim 17, further comprising a step selected from: incubating themono-PEG-IL-10 product in a TRIS buffer at pH 7.0 to 8.0; and treatingthe mono-PEG-IL-10 product with 0.05 to 0.4 M hydroxylamine HCl salt.20. A PEG-IL-10 prepared according to a process of claim 11.